Nuclease-Mediated Targeting With Large Targeting Vectors

• US 20130309670A1
• Filed: 04/25/2013
• Published: 11/21/2013
• Est. Priority Date: 04/25/2012
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Frendewey, David, Auerbach, Wojtek, Valenzuela, David M., Yancopoulos, George D.
• Granted Patent: US 9,834,786 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C07K 14/7155 for interleukins [IL]
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/907 in mammalian cells

Abstract

Compositions and methods are provided for making one or more targeted genetic modifications at a target genomic locus by employing homologous recombination facilitated by single or double-strand break at or near the target genomic locus. Compositions and methods for promoting efficiency of homologous recombination between an LTVEC and a target genomic locus in prokaryotic or eukaryotic cells using engineered nucleases are also provided.

Claims

1. A method for modifying a target genomic locus in a mammalian cell, comprising:

(a) introducing into a mammalian cell;

(i) a nuclease agent that makes a single or double-strand break at or near a target genomic locus, and

(ii) a large targeting vector (LTVEC) comprising an insert nucleic acid flanked with an upstream homology arm and a downstream homology arm; and

(b) selecting a targeted mammalian cell comprising the insert nucleic acid in the target genomic locus.

2. The method of claim 1, wherein the mammalian cell is a pluripotent cell.

3. The method of claim 1, wherein the mammalian cell is an induced pluripotent stem (iPS) cell.

4. The method of claim 2, wherein the pluripotent cell is a mouse embryonic stem (ES) cell.

5. The method of claim 2, wherein the pluripotent cell is a hematopoietic stem cell.

6. The method of claim 2, wherein the pluripotent cell is a neuronal stem cell.

7. The method of claim 1, wherein the mammalian cell is a human fibroblast.

8. The method of claim 1, wherein the mammalian cell is a human cell isolated from a patient having a disease, and wherein the human cell comprises at least one human disease allele in its genome.

9. The method of claim 8, wherein integration of the insert nucleic acid into the target genomic locus replaces the at least one human disease allele in the genome.

10. The method of claim 1, wherein combined use of the LTVEC with the nuclease agent results in an increased targeting efficiency compared to use of the LTVEC alone.

11. The method of claim 10, wherein the targeting efficiency of the LTVEC is increased at least 2-fold compared to use of the LTVEC alone.

12. The method of claim 1, wherein the nuclease agent is an expression construct comprising a nucleic acid sequence encoding a nuclease, and wherein the nucleic acid is operably linked to a promoter active in the mammalian cell.

13. The method of claim 1, wherein the nuclease agent is an mRNA encoding a nuclease.

14. The method of claim 12, wherein the nuclease is a zinc finger nuclease (ZFN).

15. The method of claim 12, wherein the nuclease is a Transcription Activator-Like Effector Nuclease (TALEN).

16. The method of claim 12, wherein the nuclease is a meganuclease.

17. The method of claim 1, wherein a target sequence of the nuclease agent is located in an intron, exon, a promoter, a promoter regulatory region, or an enhancer region in the target genomic locus.

18. The method of claim 1, wherein a sum total of the upstream homology arm and the downstream homology arm is at least 10 kb.

19. The method of claim 1, wherein the insert nucleic acid ranges from about 5 kb to 300 kb in length.

20. The method of claim 1, wherein the insert nucleic acid comprise a selection cassette.

21. The method of claim 1, wherein the insert nucleic acid comprises a reporter gene.

22. The method of claim 1, wherein integration of the insert nucleic acid into the target genomic locus results in a deletion of an endogenous gene at the target genomic locus.

23. The method of claim 1, wherein integration of the insert nucleic acid into the target genomic locus results in an addition of an exogenous sequence at the target genomic locus.

24. The method of claim 1, wherein integration of the insert nucleic acid into the target genomic locus results in a knockout, a knock-in, a point mutation, a domain swap, an exon swap, an intron swap, a regulatory sequence swap, a gene swap, or a combination thereof.

25. The method of claim 1, wherein the insert nucleic acid comprises a human nucleic sequence.

26. The method of claim 25, wherein the insert nucleic acid comprises an unrearranged human immunoglobulin heavy chain variable region nucleic acid sequence operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence.

27. The method of claim 26, wherein the immunoglobulin heavy chain constant region sequence is a mouse immunoglobulin heavy chain constant region sequence, a human immunoglobulin heavy chain constant region sequence, or a combination thereof.

28. The method of claim 26, wherein the immunoglobulin heavy chain constant region nucleic acid sequence is selected from a C_H1, a hinge, a C_H2, a C_H3, and a combination thereof.

29. The method of claim 1, wherein the insert nucleic acid comprises an unrearranged human ε

or κ

light chain variable region nucleic acid sequence operably linked to a mouse or human immunoglobulin light chain constant region nucleic acid sequence selected from a λ

light chain constant region nucleic acid sequence and a κ

light chain constant region nucleic acid sequence.

30. The method of claim 1, wherein the insert nucleic acid comprises a rearranged human ε

or κ

light chain variable region nucleic acid sequence operably linked to a mouse or human immunoglobulin light chain constant region nucleic acid sequence selected from a λ

light chain constant region nucleic acid sequence and a κ

light chain constant region nucleic acid sequence.

31. The method of claim 1, wherein selecting step (b) is carried out via a modification of allele (MOA) assay.

32. The method of claim 1, wherein the insert nucleic acid comprises a nucleic acid sequence homologous to the nucleic acid sequence in a genome of the mammalian cell.

33. The method of claim 1, wherein the insert nucleic acid comprises a nucleic acid sequence orthologous to the nucleic acid sequence in a genome of the mammalian cell.

34. The method of claim 1, wherein the insert nucleic acid comprises a nucleic acid sequence from a different species.

35. The method of claim 1, wherein the targeted mammalian cell comprises in its genome the insert nucleic acid ranging from about 5 kb to about 300 kb.

36. A method for modifying a target genomic locus of a mammal via bacterial homologous recombination (BHR) in a prokaryotic cell, comprising:

(a) introducing into a prokaryotic cell comprising a target genomic locus of a mammal;

(i) a targeting vector comprising an insert nucleic acid flanked with a first upstream homology arm and a first downstream homology arm, and

(ii) a nuclease agent that makes a single or double-strand break at or near the target genomic locus, and

(b) selecting a targeted prokaryotic cell comprising the insert nucleic acid,

wherein the target genomic locus is present in a large targeting vector (LTVEC) comprising a second upstream homology arm and a second downstream homology arm, and

wherein the prokaryotic cell is capable of expressing a recombinase that mediates the BHR.

37. The method of claim 36, wherein the mammal is a human.

38. The method of claim 36, wherein the mammal is a rodent.

39. The method of claim 38, wherein the rodent is a mouse, a rat, or a hamster.

40. The method of claim 36, wherein combined use of the targeting vector with the nuclease agent results in increased targeting efficiency compared to use of the targeting vector alone.

41. The method of claim 40, wherein the targeting efficiency is increased at least by two-fold compared to use of the targeting vector alone.

42. The method of claim 36, wherein the prokaryotic cell is a recombination-competent strain of E. coli.

43. The method of claim 36, wherein the prokaryotic cell comprises a nucleic acid that encodes the recombinase.

44. The method of claim 36, wherein the prokaryotic cell does not comprise a nucleic acid that encodes the recombinase, and the nucleic acid that encodes the recombinase is introduced into the prokaryotic cell.

45. The method of claim 36, wherein the nuclease agent is an expression construct comprising a nucleic acid sequence encoding a nuclease, and wherein the nucleic acid is operably linked to a promoter active in the prokaryotic cell.

46. The method of claim 36, wherein the nuclease agent is an mRNA encoding a nuclease.

47. The method of claim 45, wherein the nuclease is a zinc finger nuclease (ZFN).

48. The method of claim 45, wherein the nuclease is a Transcription Activator-Like Effector Nuclease (TALEN).

49. The method of claim 45, wherein the nuclease is a meganuclease.

50. The method of claim 36, wherein a target sequence of the nuclease agent is located in an intron, exon, a promoter, a promoter regulatory region, or an enhancer region in the target genomic locus.

51. The method of claim 36, wherein a sum total of the second upstream homology arm and the second downstream homology arm is at least 10 kb,

52. The method of claim 36, wherein the insert nucleic acid ranges from about 5 kb to 300 kb in length.

53. The method of claim 36, wherein the insert nucleic acid comprise a selection cassette.

54. The method of claim 36, wherein the insert nucleic acid comprises a reporter gene.

55. The method of claim 36, wherein integration of the insert nucleic acid into the target genomic locus results in a deletion of an endogenous gene at the target genomic locus.

56. The method of claim 36, wherein integration of the insert nucleic acid into the target genomic locus results in an addition of an exogenous sequence at the target genomic locus.

57. The method of claim 36, wherein integration of the insert nucleic acid into the target genomic locus results in a knockout, a knock-in, a point mutation, a domain swap, an exon swap, an intron swap, a regulatory sequence swap, a gene swap, or a combination thereof.

58. The method of claim 36, wherein the insert nucleic acid is a human nucleic sequence.

59. The method of claim 58, wherein the insert nucleic acid comprises an unrearranged human immunoglobulin heavy chain variable region nucleic acid sequence operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence.

60. The method of claim 59, wherein the immunoglobulin heavy chain constant region sequence is a mouse immunoglobulin heavy chain constant region sequence, a human immunoglobulin heavy chain constant region sequence, or a combination thereof.

61. The method of claim 59, wherein the immunoglobulin heavy chain constant region nucleic acid sequence is selected from a C_H1, a hinge, a C_H2, a C_H3, and a combination thereof.

62. The method of claim 36, wherein the insert nucleic acid comprises an unrearranged human ε

or κ

light chain variable region nucleic acid sequence operably linked to a mouse or human immunoglobulin light chain constant region nucleic acid sequence selected from a λ

light chain constant region nucleic acid sequence and a κ

light chain constant region nucleic acid sequence.

63. The method of claim 36, wherein the insert nucleic acid comprises a rearranged human ε

or κ

light chain variable region nucleic acid sequence operably linked to a mouse or human immunoglobulin light chain constant region nucleic acid sequence selected from a λ

light chain constant region nucleic acid sequence and a κ

light chain constant region nucleic acid sequence.

64. The method of claim 36, wherein selecting step (b) is carried out via a modification of allele (MOA) assay.